Pomegranate fruit polyphenol composition and methods of use and manufacture thereof

ABSTRACT

A pharmaceutical composition with an active pharmaceutical ingredient including a pomegranate fruit polyphenol extract. The pomegranate fruit polyphenol extract includes at least about 3% combined punicalagin A and punicalagin B by weight, less than about 5% ellagic acid and their derivatives by weight, and less than about 1% anthocyanins by weight.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/299,356, filed on Nov. 17, 2011, which is acontinuation-in-part of U.S. patent application Ser. No. 12/564,878,filed on Sep. 22, 2009, which is a continuation application of U.S.patent application Ser. No. 11/137,248, filed on May 24, 2005, nowissued as U.S. Pat. No. 7,611,738, all of which are herein incorporatedby reference for completeness of disclosure; the present application isalso a continuation-in-part of U.S. patent application Ser. No.11/745,440, filed on May 7, 2007, which claims priority to U.S.Provisional Application Ser. No. 60/888,763, and U.S. ProvisionalApplication Ser. No. 60/888,762, both filed Feb. 7, 2007, and which is acontinuation-in-part of U.S. patent application Ser. No. 11/687,480, nowissued as U.S. Pat. No. 7,943,185, all of which are herein incorporatedby reference for completeness of disclosure.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The disclosure provided herein relates generally to pomegranateextracts. More particularly, but not by way of limitation, one or moreembodiments enable oral or enteral dosage forms containingphytochemicals from pomegranate in a quantity reflecting that of thenatural fruit itself.

2. Description of the Related Art

The pomegranate is acclaimed for its health benefits and for itsdisease-fighting antioxidant potential. Antioxidants are importantbecause they are believed to protect the body against free radicals, theharmful molecules that can cause heart disease, premature aging,Alzheimer's disease, blindness, and a variety of cancers.

There are many kinds of antioxidants, some produced by the body andothers derived from the foods we eat. When the body's naturalantioxidant defenses are lowered, or greater amounts of free radicalsare produced, the body becomes more dependent upon food sources ofantioxidants. The consumption of phytochemical-rich diet is associatedwith a reduced risk of chronic human illnesses such as certain types ofcancers, inflammation, and cardiovascular and neurodegenerativediseases.

Studies show pomegranate juice has more polyphenol antioxidants than anyother drink, such as red wine, green tea, blueberry juice, cranberryjuice and orange juice. Two common ways of consuming pomegranates are byeating the fleshy arils of the pomegranate fruit itself and by drinkingthe juice obtained from the arils.

There are studies illustrating the beneficial effects of pomegranatephytochemicals, including polyphenols, proanthocyanidins, hydrolysabletannins, etc. Hence it is desirable to gain whatever beneficial effectsmight be present by consuming pomegranate and its phytochemicals. Theoral route is the least invasive, most convenient route foradministering pomegranate phytochemicals on a routine basis. However,the pomegranate fruit is a difficult fruit to consume and certainpomegranate phytochemicals may lose their health beneficial effects byundergoing chemical reactions into less bioavailable and/or lessbioactive forms during processing and storage of juices and extracts.

For example, a major polyphenol antioxidant called punicalagin can byhydrolyzed into ellagic acid. Once punicalagin hydrolyzes into ellagicacid its ability to offer antioxidant potency to the body is reducedsince free ellagic acid is not as bioavailable. When punicalagins arepreserved in their original unhydrolyzed and then consumed, they can beabsorbed into the bloodstream, and greater health benefits can beobtained. Punicalagins are 100% water-soluble, highly bioavailable, andshown to possess a high absorption rate up to 95%. Not only dopunicalagins offer antioxidant activity on their own, they can break upinto smaller polyphenols that are also absorbed into the body.Punicalagins are one important component of pomegranate polyphenols, butthe total composition of the polyphenols themselves is a complex mixtureof numerous other components.

Predominant types of pomegranate polyphenolic compounds are hydrolyzabletannins, which are found in the peels (rind, husk, or pericarp),membranes, and piths of the fruit. Hydrolyzable tannins, includingpunicalagin, are susceptible to enzymatic and non-enzymatic hydrolysis.Other hydrolyzable tannins are include gallic acid and ellagic acidesters of core molecules that consist of polyols, such as sugars. Duringhydrolysis, gallotannins yield gallic acid and glucose whileellagitannins yield ellagic acid and glucose. The reported solublepolyphenol content in pomegranate juice varies within the limits ofapproximately 0.2% to approximately 1.5%, and ellagic acid was measuredin commercial juices around 100 to around 3000 mg/L.

For the reasons above, many of products claiming to contain “naturalpomegranate” may in fact have less concentrated key ingredients orphytochemicals that have specific health benefits. Hence there is a needto find ways to concentrate pomegranate phytochemicals, includingpolyphenol antioxidants such as punicalagins and its automers, in theirbioavailable and bioactive.

For the reasons above, many of products claiming to contain “naturalpomegranate” may in fact lack key ingredients or phytochemicals thathave specific health benefits. Hence there is a need to find ways toconcentrate pomegranate phytochemicals, including polyphenolantioxidants such as punicalagins and its automers, in theirbioavailable and bioactive forms.

For at least the reasons described above there is a need for processesfor producing an oral or enteral dosage form containing keyphytochemicals from pomegranates.

SUMMARY OF THE INVENTION

One or more embodiments of the pomegranate fruit polyphenol extractdescribed herein are directed to a pharmaceutical composition includingan active pharmaceutical ingredient. The active pharmaceuticalingredient includes a pomegranate fruit polyphenol extract including atleast about 3% combined punicalagin A and punicalagin B by weight, andless than about 5% ellagic acid and their derivatives by weight, andless than about 1% anthocyanins by weight.

The pharmaceutical composition further includes a pharmaceuticallyacceptable carrier in instances where a carrier is desired.

In one or more embodiments of the pharmaceutical composition, thepomegranate fruit polyphenol extract may include between about 3% toabout 8% combined punicalagin A and punicalagin B by weight. Forexample, the pomegranate fruit polyphenol extract may include betweenabout 3% to about 4% combined punicalagin A and punicalagin B by weight.In another instance, the pomegranate fruit polyphenol extract maycontain between about 4.5% to about 8% combined punicalagin A andpunicalagin B by weight. In one or more embodiments, the pomegranatefruit polyphenol extract includes at least about 20% combinedpunicalagin A and punicalagin B by weight.

In one or more embodiments of the pharmaceutical composition, thepomegranate fruit polyphenol extract includes at least about 15%combined punicalagin and punicalin by weight. The pomegranate fruitpolyphenol extract may include at least about 20% combined punicalaginand punicalin by weight. Furthermore, one or more embodiments mayinclude at least about 25% combined punicalagin and punicalin by weight

The pomegranate fruit polyphenol extract may include less than about 4%ellagic acid and their derivatives by weight.

In one or more embodiments of the pharmaceutical composition, thepomegranate fruit polyphenol extract is formulated to include less thanabout 3.5% free ellagic acid by weight. Furthermore, pomegranate fruitpolyphenol extract may be formulated to include less than about 1.5%free ellagic acid by weight. In one or more embodiments, the pomegranatefruit polyphenol extract includes less than about 0.3% free ellagic acidby weight.

In one or more embodiments of the pharmaceutical composition, thepomegranate fruit polyphenol extract includes less than about 0.1%anthocyanins by weight.

In one or more embodiments, the pomegranate fruit polyphenol extract isobtained from pomegranates of the Wonderful variety but other varietalsof pomegranate fruit may be equally sufficient. The pomegranate fruitpolyphenol extract may be obtained from pomegranate solids including oneor more of the pericarp, inner membrane, and seeds.

The pomegranate fruit polyphenol extract can be delivered in variousforms and in at least one embodiment includes at least about 1000 mg ofa dry composition containing at least about 80% total polyphenols, atleast about 85% total polyphenols, or at least 90% total polyphenols.

The pharmaceutical composition can be administered in various forms may,for example, be selected from an intermediate release composition, adelayed release composition, an extended release composition, a mixedrelease composition and an enterically coated composition. Thepharmaceutical composition may be administered in a capsule or otherdosage forms such as a tablet, including but not limited to a bilayertablet. The pharmaceutical composition can also be administered orally,intramuscularly, intravenously, transdermally, buccally or topically.

DETAILED DESCRIPTION

A pomegranate fruit polyphenol extract will now be described. In thefollowing exemplary description, numerous specific details are set forthin order to provide a more thorough understanding of the embodimentsdescribed throughout this disclosure. It will be apparent, however, toan artisan of ordinary skill in the art that the present invention maybe practiced without incorporating all aspects of the specific detailsdescribed herein. In other instances, specific features, quantities,methods, or measurements well known to those of ordinary skill in theart have not been described in detail so as not to obscure theinvention. Readers should note that although examples of the inventionare set forth herein, the claims, and the full scope of any equivalents,are what define the scope of the invention.

As used herein, the term “phytochemicals” refers collectively tocompounds which are naturally-occurring in the pomegranate and toreaction products and metabolites of these compounds, which areconsidered to have a beneficial effect on the human or animal health.Examples of such phytochemicals include, but not limited to phenolics,polyphenols, and phenolic acids, sterols and triterpenoids, fatty acidsand triglycerides, and alkaloids.

As used herein, the term “polyphenols” refers generally to a family ofnaturally-occurring compounds in the pomegranate and includes phenolsand polyphenols. Phenols are a class of chemical compounds consisting ofa single phenol unit in their structure. Although similar to alcohols,phenols have unique properties including relatively higher acidities dueto the aromatic ring tightly coupled to the oxygen and a relativelyloose bond between the oxygen and the hydrogen. Examples of phenoliccompounds within this group include ellagic acid and gallic acid.Polyphenols are a group of compounds, characterized by the presence ofmore than one phenolic group. Polyphenols include tannins (e.g.,ellagitannins and gallotannins), flavonoids (e.g., anthocyanins andisoflavones) and stilbenes (e.g., resveratrol).

As used herein, the term “pomegranate juice” refers to the juice that issubstantially obtained from the arils of the pomegranate.

As used herein, the term “pomegranate solids” refers to any one or acombination of the pericarp, the inner membrane and seeds of apomegranate.

The pomegranate fruit polyphenol extracts described herein areformulated for pharmaceutical or nutriceutical purposes and highlybio-available. In one or more embodiments, such pomegranate fruitpolyphenol extracts comprise a significantly higher total polyphenolcontent, particularly of the high molecular weight polyphenol (e.g.,punicalagin), than is found in pomegranate juice. The pomegranate fruitpolyphenol extracts can be administered in various forms and may bemixed with food products, beverages and/or pharmaceutical compositionsthat complement or increase the effectiveness of the pomegranate fruitpolyphenol extract.

One or more embodiments of pomegranate fruit polyphenol extracts areincluded in a pharmaceutical composition. Such pharmaceuticalcompositions may be in form of tablets, suspensions, implants,solutions, emulsions, capsules, powders, syrups, liquid compositions,ointments, lotions, creams, pastes, and gels. Such pharmaceuticalcompositions may also be in form of pharmaceutical preparations,nutritional supplements, vitamin supplements, food additives, and foodsupplements.

The pomegranate fruit polyphenol extracts may also be included in apharmaceutical composition along with one or more pharmaceuticallysuitable carriers. Suitable carriers or excipients are inert ingredientsand can be included in the composition. Excipients can include, but arenot limited to, fillers sugar alcohols, starch, lubricants, and binders.Examples of sugars include lactose, glucose, and sucrose. Sugar alcoholsinclude mannitol, sorbitol, and xylitol. Examples of starch includewheat, corn, or potato starch, modified starch and sodium starchglycolate. Lubricants include talc, magnesium stearate, calciumstearate, colloidal silica, and stearic acid. Binders includepolyvinylpyrrolidone, cellulose derivatives, carboxymethyl cellulose,hydroxylpropyl cellulose, hydroxypropylmethyl cellulose, methylcellulose, and gelatin. Conventional procedures for preparing suchcompositions in appropriate, dosage forms of the extract may beutilized. Such compositions may be administered orally or parenterallyemploying liquid form preparations containing the extract. Thecompositions may be administered orally, in appropriate dosage units ofthe extract in a pharmaceutically acceptable carrier or excipient. Thus,the compositions may be formulated into solid or liquid preparations,such as capsules, pills, tablets, powders, solutions, suspension, oremulsions and prepared according to methods known in the art for themanufacture of such compositions. The solid unit dosage forms may be inform of a hard or soft shelled gelatin capsule containing the extractand a suitable carrier or excipient.

The composition may also be administered parenterally as injectabledosages in a physiologically acceptable carrier. Parenteraladministration may be subcutaneous, intravenous, intramuscular, orinterperitoneally.

One or more embodiments of compositions containing the extract and thepomegranate juice are provided. The combination of the extract andpomegranate juice not only produces a composition having a higher totalpolyphenol content, as compared to the pomegranate juice alone, but italso provides the broad spectrum of the different polyphenols whichpredominate the pomegranate juice and extract. The pomegranate fruitpolyphenol extracts are provided for preventing or ameliorating diseaseconditions in a subject by administering to the subject an effectiveamount of the composition suitable for use in pharmaceutical compositionor nutritional preparation. Such disease conditions includepolyphenol-mediated diseases and cancer. Examples of polyphenol-mediateddiseases include circulatory disorders such as hypertension and coronaryartery disease, erectile dysfunction, lung disorders such as asthma,cancers of various types, inflammatory conditions, certain liverconditions, diabetes, mood disorders, eye disorders such as cataracts,weak eyesight due to aging, macular degeneration, and other age-relateddisorders, such as Alzheimer's disease and dementia.

Examples of methods are provided for modulating the growth andprogression of cancerous cells, the methods comprising selecting asubject having cancerous cell growth and administering to the subject aneffective amount of the composition containing the extract. One or moreembodiments described are provided for preventing or slowing increasesin the Prostate Specific Antigen (PSA) levels in a subject havingprostate cancer. An effective amount of a pharmaceutical compositioncomprising pomegranate fruit polyphenol extracts is administered to asubject having prostate cancer.

One or more embodiments of pharmaceutical compositions including thepomegranate fruit polyphenol extracts described herein include aneffective amount of pomegranate fruit polyphenol extract sufficient toachieve the intended beneficial health results. Accordingly, theeffective amount of the composition to be administered depends onconsiderations such as the dosage unit employed, the mode ofadministration, the period of treatment, the age, sex and weight of theperson treated and the nature and extent of the condition treated. Theeffective amount can readily be determined based upon standardtechniques known to evaluate whether the intended effect of thecomposition has been achieved, by standard toxicity tests and bystandard pharmacological assays.

Pomegranate Polyphenols

Turning to pomegranate extracts, it was been surprisingly discoveredthat extracts obtained from the pomegranate solids, in accordance withthe methods disclosed herein, have substantially higher total polyphenolcontent than is found in the juice from the pomegranate arils. This isparticularly true with respect to the higher molecular weightpolyphenols (e.g., punicalagin).

Punicalagin is a powerful antioxidant, which is found in at least twoisoforms (punicalagin-α and punicalagin-β). Two features of punicalaginsrelate to the protection of cardiovascular function and accuratecellular replication. Thus, the punicalagin automers are responsible, inpart, for the high antioxidant activity of the extract. While theantioxidant and other beneficial health effects of the extract are dueto the presence of polyphenols, the presence of other phytochemicalcompounds in the extract, or the synergistic effect of thesephytochemicals, can also be responsible for the antioxidant and otherbeneficial health effects of the extract.

In addition to punicalagin, other high molecular weight polyphenols havebeen characterized in the extract of pomegranate solids. These highmolecular weight polyphenols include ellagitannin and other hydrolysabletannins, such as punicacortein A, punicalin, pedunculagin, andgallotanin dimers and trimers. The pomegranate extract in theadministered composition can be between about 20% to about 40% by HPLCarea of a combination of punicalin and punicalagin; and between about 1%to about 5% by HPLC area of ellagic acid. In one or more embodiments,the punicalagin includes the automers punicalagin-α and punicalagin-β.composition can also comprise other pomegranate polyphenol compounds,including, but not limited to, gallic acid, an isoflavone (e.g.,genistein or daidzein, as well as methylated pr glycoside derivativesthereof), an anthocyanin, a hydrolyzable tannin, and/or combinationsthereof.

Moreover, a large number of anthocyanins have been characterized in theextract of the pomegranate solids. Examples of the anthocyanins includepelargonidin 3-glucoside, cyaniding 3-glucoside, delphinidin3-glucoside, pelargonidin 3,5-diglucoside, cyaniding 3,5-diglucoside,and delphinidin 3,5-diglucoside. Although these anthocyanins have beencharacterized in both the pomegranate juice and the extract, these lowermolecular weight polyphenols comprise a higher proportion of the totalpolyphenol content in pomegranate juice (approximately 50%) than in theextract.

Pomegranate Fruit Polyphenol Extracts and Extraction Methods

The pomegranate fruit polyphenol extracts are obtained from at least onepomegranate solid. For instance, one may make use of the pericarp, innermembrane and seeds to create a mixture comprising the pomegranate solidsin an aqueous solution. In one or more embodiments, the mixture of thepomegranate solids may be created by adding water in an amount that isabout 20 to about 80% w/v, and more preferably about 50% w/v, of thepomegranate solids. The mixture is preferably crushed or milled tocreate a rough grind of pomegranate solids dispersed in the aqueoussolution.

In other instances the starting materials for production of thepomegranate fruit polyphenol extract are solids from the husks andresidual fruits remaining after the first or second pressing of wholefruits in the production of the juice concentrate. Both powder orconcentrated liquid forms may be made with these materials. In one ormore embodiments, the mixture of the solids may be created by addingwater in an amount that is about 20 to about 80% w/v, and morepreferably about 50 w/v, of the solids. The mixture may be crushed ormilled to create a rough grind of pomegranate solids dispersed in theaqueous solution.

The extracts obtained from pomegranate solids with a substantiallyhigher total polyphenol content than is found in the juice from thepomegranate arils. This is particularly true with respect to the highermolecular weight polyphenols and, in particular, punicalagin.

In one or more embodiments, the pomegranate fruit polyphenol extract isproduced by providing pomegranate solids selected from the groupconsisting of the pericarp, inner membrane and seeds and creating amixture comprising the pomegranate solids in an aqueous solution. In anexemplary embodiment, the mixture of the pomegranate solids is createdby adding water in an amount that is about 20 to about 80% w/v, and morepreferably about 50% w/v, of the pomegranate solids. The mixture ispreferably crushed or milled to create a rough grind of pomegranatesolids dispersed in the aqueous solution. The mixture is then heated toa temperature of about 60° F. to about 210° F., about 85° F. to about185° F., or about 110° F. to about 160° F. The temperature to which themixture is heated depends upon the selection of enzymes, or combinationof enzymes, added to the mixture. In one or more embodiments, themixture is heated to a temperature that permits the maximum catalysis ofthe enzyme or combination of enzymes. Enzymes may also be added beforethe mixture is heated. Thus, the order of the steps of heating themixture and adding the enzymes is not critical, so long as the mixtureis heated to a temperature that permits the enzymes to at leastpartially degrade the pomegranate solids and liberate phytochemicalsfrom the plant tissues and/or cells. Once liberated, the phytochemicalsmay react and/or polymerize to create new phytochemical compounds orreaction products. In one or more embodiments, anthocyanins are degradedduring the treatment process.

Enzymes suitable for use in accordance with this embodiment includethose capable of at least partially degrading the plant tissue or cellsto liberate the phytochemicals from the pomegranate solids. Such enzymesinclude any one or a combination of pectinase, cellulase, hemicellulase,amylase, arabanase, and other hydrolyzing enzymes, to name a few. Theenzymes added to the mixture may be naturally-occurring or synthetic.They may be derived from any one or a combination of sources, such asanimal, plant, fungal, and bacterial sources. The amount of the enzymeor combination of enzymes added to the mixture depends on thetemperature of the mixture and the amount of pomegranate solids presentin the mixture. After the enzymes have at least partially degraded thepomegranate solids, the residual insoluble solid materials, such asproteins, are removed from the mixture. Optionally, a clarificationagent, such as bentonite, may be added before the step of removing theresidual insoluble materials from the mixture. The removal of residualinsoluble materials from the mixture may be accomplished by filtration,centrifugation, chromatographic techniques, and other techniques. Inanother embodiment of the invention the mixture of pomegranate solids isheated to liberate the polyphenols and enzymes are not utilized.

The pomegranate fruit polyphenol extract may be produced bymicro-filtration. In one or more embodiments, a molecular weight cut-offof at least 1,000 Da is used. Alternatively, a molecular weight cut-offof between about 4,500 Da to about 5,500 Da is used. In one or moreembodiments, micro-filtration is performed using a membrane filter ratedfor up to about 1 μm. The resulting liquid extract may be concentratedin an evaporator under vacuum to about 50 tou about 90 Brix (Bx),preferably to about 60 to about 80 Bx, and more preferably to about 70Bx, more preferably to about 65 Bx to about 70 Bx. The extract can thenbe and pasteurized at a temperature and for a length of time sufficientto kill microorganisms that could cause disease, spoilage or undesiredfermentation. In one preferred embodiment, the extract may bepasteurized at a temperature of about 140° F.-280° F., preferably ofabout 195° F.-240° F., and optimally of about 205° F. The pasteurizationmay also denature the remaining enzymes that were added to the mixture.

Polyphenols are recovered from the extract concentrate using FDAfood-grade resins. Extract concentrate, diluted with water, is passedthrough resin columns which preferentially adsorbs polyphenols from theextract liquid. The resins do not chemically modify the polyphenols;they reversibly adsorb and desorb the polyphenols with their originalchemical structure remaining unchanged. Non-phenol compounds, such assugars, organic acids, cellulose, and other carbohydrates, passunadsorbed through the resin column.

Polyphenols adsorbed on the resin are recovered (de-adsorbed) by elutionusing ethanol in water. In one or more embodiments, the recoveredpolyphenols include substantially less anthocyanins than the extractliquid, at least partially due to loss during adsorption and/or elution.The recovered polyphenols are concentrated by completely removingethanol. The remaining polyphenol water solution is dried to produce apomegranate food polyphenol extract powder. The separation medium mayinclude a synthetic polymeric adsorbent resin. Generally these syntheticpolymeric adsorbents take the form of non-ionic macroreticular resinsthat adsorb and release ionic and polar molecules (compounds) throughhydrophobic and polar interactions; these are usually employed underisocratic conditions (i.e., only a single eluent of fixed composition isused). Such polymeric resins are usually derived from a synthetichydrophobic polyaromatic resin such as cross-linked polyvinylbenzene(polystyrene) and polydivinylbenzene. These resins are manufacturedunder trade names such as Amberchrom™, Amberlite™, Diaion™, and Dowex™.One advantage of the polystyrene-divinylbenzene copolymer resin is thepolyphenols are especially well adsorbed when dissolved in water ordilute aqueous C1-C3 alkanol (e.g. 2% v/v ethanol), preferably at thenominal operating range of 100° to 140° F. It may be possible to usenatural polymeric media, such as microparticulate cellulose which isparticularly well suited for the separation of nucleotides, sugars,amino acids and polyphenols. Potential drawbacks to the use ofmicroparticulate cellulose or derivatives thereof, are swelling in anaqueous environment and/or compressibility under pressure. Otheralternatives are dextran polymers (e.g. Sephadex™, Pharmacia UK) oragarose beads (e.g., Sepharose™, Pharmacia, UK). In one or moreembodiments, temperature range for operating the separation process isin the range of 100° to 140° F. Less preferred is using the separationmedium at the temperature below 100° F. which alters the adsorptioncharacteristics of polyphenols and other phytochemicals of interest. Inone or more embodiments, the flow rate through the separation medium isin the range one to three bed volumes an hour, optimally two bed volumesper hour. A bed volume is the amount of the adsorbent resin in aseparation medium. The volume and total time of flow of a particularfeed stream into the column can be controlled by the desired output andthe input stream. The optimal flow rate enables the separation medium tosequester polyphenols and other phytochemicals of interest, and rinseout the unbound material.

For the rinsing step in one or more embodiments, a dilute aqueousalcohol is passed through the separation medium to remove unboundmaterial (e.g., sugars, proteins, fibers, enzymes, carbohydrates). Whenobtaining a high purity of polyphenols in the pomegranate drycomposition is desired, it is preferred that this rinsing step beperformed prior to the elution of polyphenols. Optionally, the rinsestep may include back flushing the separation medium with a diluteaqueous alcohol to remove any insoluble material that may collect on thetop of the separation medium. Dilute aqueous alcohol is any aqueoussolution containing alkanol having one to four carbon atoms and of lessthan about 5% v/v, more preferable 2% v/v. Ethanol is the preferredalkanol since it is approved for food use, although other alkanols maysuffice. Less preferable is water, which is not as effective at gettingunbound material out of the separation medium and reduces the purity ofpolyphenols in the pomegranate dry composition.

In one or more embodiments which is described herein for purposes ofexample, the pomegranate fruit polyphenol extract is produced byobtaining pomegranate solids, which generally comprise the pericarp, theinner membrane and seed of the pomegranate. In one or more exemplaryembodiments, the pomegranate solids are obtained and collected after theprimary juice from the arils is substantially expelled or otherwiseremoved from the pomegranate by pressing, crushing, or other methodsknown to the art for extracting pomegranate juice. The pomegranatesolids are then transferred to three Reitz mills (such as those sold byReitz Mills) with ⅜ inch screens. The pomegranate solids are milled to afine puree and heated to approximately 125° F. This step, coupled withthe following enzyme addition, assists in breaking down the colloidalstructure of the remaining pomegranate solids, thereby releasing theremaining soluble solids. The mixture is heated to a temperature ofabout 125° F. for two hours. In one or more of the exemplary embodimentsdescribed here three enzymes are added to the mixture: pectinase(Rohapect® DA6L), cellulase/pectinase (Rohapect® CL), andhemi-cellulase/pectinase (Rohapect® B1L). These enzymes liberate theremaining pomegranate soluble solids, such as sugars, minerals,anthocyanins, and remaining polyphenols. The mixture is then pumped fromthe extraction plant to the primary processing plant where it is held inmash treatment tanks for approximately one hour. After one hour, 50-100pounds of bentonite in a 125 gallon water slurry, per 8,000 gallons ofthe mixture, is added for protein removal. The treated mixture was thenpassed through a Westphalia 755 Decanter for removal of solids. Theresidual insoluble material is typically discharged as waste. The liquidextract then exits the decanter and is filtered on on microfiltrationmembranes (such as one sold by Koch SUPER-COR®) at a 500,000 Damolecular weight cut-off and then filtered again on Koch ultrafiltrationmembranes at a 100,000 or 200,000 Da molecular weight cut-off. In one ormore embodiments, the filtered liquid extract is optionally applied to arising-film plate evaporator (such as that sold by Schmidt-Bretten.Initial heat on this step is about 140° F. In this step, the filteredliquid extract is concentrated to about 15° to 20° Bx. The filteredliquid extract is maintained at the temperature of about 140° F. andthen passed through a pre-heated preparative column at about 140° F.(4-foot diameter, 4-foot tall) packed with Amberlite™ FPX66 (Rohm andHaas, Philadelphia, Pa.) at the flow rate of about two bed volumes anhour until the resins gets loaded. Any portions of liquid effluentindicating a bleed-through of polyphenols may be collected forsubsequent loading step. After the load step, dilute aqueous alcohol (2%ethanol/H₂O) is passed through the preparative column at the flow rateof about two bed volumes an hour to remove unbound material. Diluteaqueous alcohol effluent is discarded as a waste. After the rinse step,concentrated aqueous alcohol (20% ethanol/H₂O) is applied to the resinand the liquid eluate containing polyphenols is collected.

Pomegranate Dry Composition

As set forth throughout this disclosure the pomegranate fruit polyphenolextract takes the form of a dry pomegranate composition in at least oneor more embodiments of the invention. This dry pomegranate compositionis a lyophilized, or freeze-dried pomegranate fruit polyphenol extract.The dry pomegranate composition may also be obtained by applying heat,using desiccants, reducing pressure, applying air, or any otherevaporative process capable of removing moisture, including anycombination thereof.

In addition to punicalagin, other high molecular weight polyphenolscharacterized in the dry pomegranate composition include ellagitanninand other hydrolysable tannins, such as punicortein A, punicalin,pedunculagin, and gallotannin dimers and trimers. Once obtained the drycomposition may be filled into capsules for distribution in pill form ormixed into various liquids to increase the polyphenol content of aparticular liquid.

In one or more embodiments, the pomegranate dry composition is obtainedfrom the pomegranate solid, in accordance with the methods disclosedherein. For instance, one may make use of the pericarp, inner membraneand seeds to create a mixture comprising the pomegranate solids in anaqueous solution. The mixture of the pomegranate solids may be createdby adding water in an amount that is about 20 to about 80% w/v, and morepreferably about 50% w/v, of the pomegranate solids. The mixture ispreferably crushed or milled to create a rough grind of pomegranatesolids dispersed in the aqueous solution. Once obtained the drypomegranate composition may be filled into capsules for distribution inpill form or mixed into various liquids to increase the polyphenolcontent of a particular liquid.

In one or more embodiments, the dry pomegranate composition includes atleast about 20-25% combined punicalin and pulicalagin by HPLC area %.Table 3 includes 3 exemplary products manufactured with different rangesof punicalagin A and punicalagin B by weight.

In one or more embodiments, the dry pomegranate composition includesless than about 5% ellagic acid and their derivatives by HPLC area %. Inone or more embodiments, the dry pomegranate composition furtherincludes less than about 4% ellagic acid and their derivatives byweight. In one or more embodiments, the dry pomegranate compositionincludes less than about 3% ellagic acid and their derivatives byweight.

The dry pomegranate composition further includes up to a trace amount ofanthocyanins. The trace amount may be about 1% anthocyanins by weight,but depending on the method of manufacture, can less than about 0.1%anthocyanins by weight. In one or more embodiments, the trace amount ofanthocyanins may be close to about 0%. Table 1 shows a product whereanthocyanins were virtually undetectable using an HPLC analysis.

In one or more embodiments, the dry pomegranate composition includes atleast about 85% total polyphenols. In other embodiments, the drycomposition includes at least about 90% total polyphenols. In one ormore embodiments, the starting materials for production of the drypomegranate composition are solids from the husks and residual fruitsthat remain after the first or second pressing of whole fruits in theproduction of the juice concentrate. Both powder or concentrated liquidforms may be made with these materials. In one or more embodiments, themixture of the solids may be created by adding water in an amount thatis about 20 to about 80% w/v, and more preferably about 50 w/v, of thesolids. The mixture may be crushed or milled to create a rough grind ofpomegranate solids dispersed in the aqueous solution.

Punicalagins and other phytochemicals of pomegranate may remain stablewhen processed and stored in the pomegranate dry composition. Thus, alower amount of the hydrolysable tannins, for example, in the drycomposition become hydrolyzed to nonbioavailable and/or less bioactiveforms. Thus one or more embodiments may be used in oral or enteraldosage form as a pharmaceutical or nutraceutical composition including,for instance, a pomegranate dry composition. This composition may beadministered in oral, pill or liquid form.

The resulting liquid eluate containing polyphenols is turned into thedry pomegranate composition by employing any suitable drying methodologyand apparatus. Drying can be accomplished, for example, by use of arotary evaporator under reduced pressure and followed by further dryingin a desiccator. Another example of a drying methodology islyophilization, which comprises freezing the liquid extract and thendrying the same under high vacuum conditions in order to allow the waterin the solid state to sublimate at low temperature. That is, the wateris removed from the material by passing directly from the solid to thegaseous state, without passing through the liquid state. One or moreembodiments utilize tray drying or spray drying the liquid eluate toformulate the pomegranate fruit polyphenol extract.

In one or more embodiments, the pomegranate dry composition may beformulated in nutraceutical compositions and be delivered in an oral orenteral dosage form. Such compositions may be administered orally orenterally employing dry form preparations containing the pomegranate drycomposition. The preparation of the nutraceutical composition mayoptionally include one or a combination of suitable binders, carriers,disintegrants, excipient, lubricants, colorants, and diluents. Suchnutraceutical composition optionally comprises one or more additionalcoatings surrounding the core and/or the control releasing coat such asmoisture barrier coats, enteric coats or coatings that affect thephysical integrity and/or appearance of the nutraceutical composition.

The dosage used in the embodiments may be applied in any suitable form,such as bars, pills, capsules, gels, liquid, etc. A dosage unit maycomprise a powder, solid or semisolid form, and more acceptable in adosage form includes without limitation, caplets, capsules, gelatincoated capsule, granules, microparticles, microspheres, pills, powder,tablets, and other solid or semisolid formulations. The solid orsemisolid dosage form preferably has a weight between 0.1 and 30 grams,more preferably between 0.2 and 10 grams. In the embodiments, a dailydosage of the prepared polyphenol can include one or more pills, tabletsor other dosage forms. Concentrated liquid forms are also contemplatedand may be made by mixing in the powder or other forms of the polyphenolcomposition described herein. Alternatively or in addition the liquidconcentrate itself can be produced from byproducts of the juiceproduction process.

According to at least one embodiment of the invention the enteral ororal administration of a nutraceutical composition includes thepomegranate dry composition. As mentioned concentrated liquid forms arealso feasible. Particularly suitable is the administration of a dosageor serving of the nutraceutical composition containing the pomegranatedry composition. Capsule form is typically most well suited for easyconsumption but all other alternatives are within the scope and spiritof the invention. The pomegranate dry composition has a substantiallyhigher polyphenol content than other known compositions. This isparticularly true with respect to the higher molecular weightpolyphenols and, in particular, punicalagins. In addition topunicalagin, other high molecular weight polyphenols characterized inthe pomegranate dry composition include ellagitannin and otherhydrolysable tannins, such as punicortein A, punicalin, pedunculagin,and gallotannin dimers and trimers. The amount of pomegranate extract tobe administered can be between about 1,000 mg to about 2,000 mg per dayand anywhere in between. Dosage may exceed 2,000 mg so long as the noobserved adverse effect level (NOAEL) is not exceeded. In humans theNOAEL is thought to be about 7,900 mg per day, assuming a 70 kg subject.

Dry Pomegranate Composition Analysis

An exemplary analysis of the dry pomegranate composition now follows.The pomegranate dry composition was analyzed using HPLC and MALDI-TOF.The pomegranate dry composition has a higher proportional content ofpomegranate polyphenols, primarily punicalagin and its automers. Table 1shows a composition breakdown in 100 grams of an exemplary pomegranatedry composition powder. Table 2 shows an HPLC analysis for an exemplarypomegranate fruit polyphenol composition. Table 3 shows a breakdown ofspecific polyphenols in three exemplary pomegranate fruit polyphenolextracts.

Once created the powder and liquid forms provide similar polyphenolcomponents of one serving of pomegranate juice. One serving ofpomegranate juice contains at least 800 mg total natural polyphenols(650 mg gallic acid equivalent, GAE) with expected variation from yearto year and batch to batch. Pomegranate juice has been shown to have upto 4,370 mg/L or 1,049 mg/8 oz of punicalagin compounds by Cerda et.al., 2004. GAE underestimates the total polyphenol level because gallicacid is not optimized standard. A 1,000 mg capsule of powder made usingthe process described herein contains at least 800 mg natural polyphenolusing a pomegranate polyphenol standard, such as but not limited to thestandard described in K. Martin et al. “Development of a novelpomegranate standard and new method for the quantitative measurement ofpomegranate polyphenols” Journal of Science of Food and Agriculture,2009; 89:157-162, which is hereby incorporated by reference in itsentirety. The table below is illustrative: About 1000 mg of the drypomegranate composition and about 200 mg of maltodextrin, which is usedas a flow agent, are intimately mixed for prior to filling in a capsulecomprising of hydroxypropyl methylcellulose.

This amount provides the polyphenol content equivalent to an 8 oz.bottle of pomegranate juice. The purification process of Example 1enables the requisite polyphenol dose in a single capsule.

TABLE 1 SPECIFICATIONS Chemical Classification Organic, NutritivePhysical Classification Dried Fruit Color Red-Brown Odor CharacteristicTannin Taste Characteristic Tannin Plant Part Used Husk, Arils (Juice),and Fruit Chemical Parameters Total Phenolics >85% (UV adsorption std.to pomegranate polyphenols) pH (1 g/100 ml water) 3.0 to 5.0 HeavyMetals (ppm) Less than 1 ppm. Physical Parameters Particle Size (wt. %retained on 60 mesh) Less than 2%. Bulk Density (g/cc) 0.8 to 0.9 TapDensity (g/cc) 0.9 to 1.0 Microbiological Assays: Total Plate Count(CFU/g) <1,000 Yeast (CFU/g)   <100 Mold (CFU/g)   <10 Total Coliforms(CFU/g)   <10 (none detected) E. coli (CFU/g)   <10 (none detected)Salmonella (CFU/g) Negative in 25 grams. Staph. aureus (CFU/g)   <10(none detected) SHELF LIFE 18 months at or below 70 F. in a sealedcontainer. PACKAGING 10 kg. in a double-lined plastic bag. Productshould be stored in the original sealed container and tightly closedafter usage.

TABLE 2 HPLC Analysis of pomegranate fruit polyphenol compositionEstimated polyphenol HPLC content from Folin % area under curve NPPE(mg/1000 mg) Monomeric HP Punicalin 3.45 0.94 1.00 27.27 7.90 8.60Monomeric HP Punicalagin A 5.70 4.75 4.79 45.02 39.90 41.19 Monomeric HPPunicalagin B 12.29 11.14 12.94 97.11 93.58 111.28 Oligomeric HPs 75.0679.23 77.10 592.98 655.53 633.06 Delphinidin 3, 5 diglucoside 0.00 0.000.00 0.00 0.00 0.00 Cyanidin 3, 5 diglucoside 0.00 0.00 0.00 0.00 0.000.00 Delphinidin 3 glucoside 0.00 0.00 0.00 0.00 0.00 0.00 Cyanidin 3glucoside 0.00 0.00 0.00 0.00 0.00 0.00 Ellagic Acids 3.50 3.94 4.1727.63 33.10 35.86 Folin Total Polyphenols 3.50 3.94 4.17 27.63 33.1035.86 Group Totals 790.00 840.00 860.00 HP (Hydrolysable polyphenols)96.50 96.06 95.83 762.37 806.90 824.14 Anthocyanins 0.00 0.00 0.00 0.000.00 0.00 Ellagic acid aglycones 3.50 3.94 4.17 27.63 33.10 35.86Non-anthocyanins (Gps 1 + 3) 100.00 100.00 100.00 790.00 840.00 860.00Punicalins + Punicalagins 21.44 16.83 18.73 169.40 141.38 161.07

TABLE 3 Method Product x1 Produt xp Product xm Component Type Nominal (%by weight) Total Hydrolyzable Tannins Gallic acid (free) HPLC, 0.05 to0.15 0.8 to 1.2 0.8 to 1.2 Ellagic acid (free) UV-vis 0.1 to 0.3 1.5 to3.5 1.5 to 2.5 Total Punicalagin A&B Punicalagin A HPLC, 0.80 to 0.951.0 to 2.0 5.5 to 6.5 Punicalagin B UV-vis 2.6 to 2.9 3.5 to 6.0 15 to16 Punicalagin A & B 3.0 to 4.0 4.5 to 8.0 20 to 22

What is claimed is:
 1. A pharmaceutical capsule comprising: apomegranate fruit polyphenol extract composition obtained frompomegranate solids comprising pericarp, inner membrane, and seeds,wherein said pomegranate fruit polyphenol extract comprises at least 3%combined punicalagin A and punicalagin B by weight, and less than 5%ellagic acid by weight.
 2. The pharmaceutical composition of claim 1,wherein said pomegranate fruit polyphenol extract comprises betweenabout 3% to about 8% combined punicalagin A and punicalagin B by weight.3. The pharmaceutical composition of claim 1, wherein said pomegranatefruit polyphenol extract comprises at least 4.5% combined punicalagin Aand punicalagin B by weight.
 4. The pharmaceutical composition of claim1, wherein said pomegranate fruit polyphenol extract comprises at leastabout 20% combined punicalagin A and punicalagin B by weight.
 5. Thepharmaceutical composition of claim 1, wherein said pomegranate fruitpolyphenol extract comprises at least about 15% combined punicalagin andpunicalin by weight.
 6. The pharmaceutical composition of claim 1,wherein said pomegranate fruit polyphenol extract comprises at leastabout 25% combined punicalagin and punicalin by weight.
 7. Thepharmaceutical composition of claim 1, wherein said pomegranate fruitpolyphenol extract comprises less than about 4% ellagic acid by weight.8. The pharmaceutical composition of claim 1, wherein said pomegranatefruit polyphenol extract comprises less than about 1.5% free ellagicacid.
 9. The pharmaceutical composition of claim 1, wherein saidpomegranate fruit polyphenol extract comprises less than about 0.3% freeellagic acid.
 10. The pharmaceutical composition of claim 1, whereinsaid composition further comprises a pharmaceutically acceptablecarrier.
 11. The pharmaceutical composition of claim 1, wherein saidpomegranate fruit polyphenol extract comprises less than about 0.1%anthocyanins by weight.
 12. The pharmaceutical composition of claim 1,wherein said pomegranate fruit polyphenol extract comprises at leastabout 1000 mg in a dry composition.
 13. A pharmaceutical capsulecomprising: a pomegranate fruit polyphenol extract composition obtainedfrom pomegranate solids comprising pericarp, inner membrane, and seeds,wherein said pomegranate fruit polyphenol extract comprises at least 3%combined punicalagin A and punicalagin B by weight, and less than 5%ellagic acid by weight; and a pharmaceutically suitable carrier.